Voltage-gated ion channels (VGC) are proteins found in the membranes of practically all cells and that through opening and closing (gating) events let ions flow through between the internal and external milieu of the cells acting as very fast signaling entities. The most characteristic and intriguing aspect of VGC is that their function is modulated by voltage. That means that the protein senses changes in the electrical field and responds by opening through a sequence of conformational changes. With the advent of high resolution electrical recording techniques combined with the molecular cloning and engineering of ion channel proteins, it has been possible to identify parts of VGC that serve as voltage-sensors. This information along with the available solved crystal structures of three VGC, has led to the proposal of several mechanistic models of voltage-sensing and how these changes are translated into channel opening. Yet, the molecular and physical natures of the events that take place during voltage-gating are not resolved and are the matter of ongoing discussion and controversy. It is the long-term goal of this proposal to contribute a physical molecular model of how VGC gate by studying intra-molecular distances at rest and while channels are open, using optical tools along with functional recordings. We will use the bacterial potassium channel, KVAP, which can be produced in large quantities in bacterial culture, purified and reconstituted into lipid membranes, which provides a unique opportunity to address these questions in molecular detail. And, we will also use the well-studied Shaker potassium channel, a mammalian muscle channel and a voltage-sensitive phosphatase for in vivo studies. The specific aims are: Aim 1. To determine in vitro in KVAP channels and in vivo in Shaker channels intra-molecular distances and their changes in response to membrane potential changes focusing on the voltage sensing domain;and, Aim 2. To extend in vivo distance measurements to other voltage-dependent membrane proteins, including a mammalian sodium channel (NaV1.4) and the Ci-VSP, a voltage-dependent phosphatase. To measure distances, a specific Lanthanide Binding Tag (LBT, that binds terbium and acts as a donor) is encoded into different parts of the protein and either another genetically encoded tag (a hexa-histidine tag) or a cysteine (to be labeled with a fluorescent probe) are introduced in another part of the same protein to act as acceptor. The terbium emits upon excitation of a nearby tryptophan residue encoded in the LBT. Because the donor and acceptor will be placed in areas suspected to participate in voltage gating, these measurements are expected to contribute real molecular distances and information on molecular rearrangements occurring during voltage gating. VGC are particularly important in nerve and muscle cells because they determine cell excitability and participate in cell-to-cell communication. The results from this work will broaden our understanding of a large number of voltage-gated proteins that are crucial in health and shall help to draw strategies to ameliorate or perhaps eventually cure some illnesses that involve the dysfunction of this important family of proteins. PUBLIC HEALTH RELEVANCE: Using state-of-the-art techniques in electrophysiology and spectroscopy (lanthanide energy transfer), combined with molecular biology, we propose here to determine in vivo the movement of crucial functional elements of membrane proteins that respond to changes in the electric field across the cell membrane. These proteins (ion channels) are found in most cells but especially in nerve and muscle cells where they determine and modulate the cells'responsiveness when challenged by a stimulus (chemical or electrical). Natural mutations in these proteins often lead to neurological and muscle related diseases known as channelopathies, therefore to overcome or cure channelopathies there is a need to understand at a molecular level how these proteins sense the environment and what changes in conformation occur during this process to understand how the system fails.